Sequence analysis in Bos taurus reveals pervasiveness of X-Y arms races in mammalian lineages.

Studies of Y Chromosome evolution have focused primarily on gene decay, a consequence of suppression of crossing-over with the X Chromosome. Here, we provide evidence that suppression of X-Y crossing-over unleashed a second dynamic: selfish X-Y arms races that reshaped the sex chromosomes in mammals as different as cattle, mice, and men. Using super-resolution sequencing, we explore the Y Chromosome of Bos taurus (bull) and find it to be dominated by massive, lineage-specific amplification of testis-expressed gene families, making it the most gene-dense Y Chromosome sequenced to date. As in mice, an X-linked homolog of a bull Y-amplified gene has become testis-specific and amplified. This evolutionary convergence implies that lineage-specific X-Y coevolution through gene amplification, and the selfish forces underlying this phenomenon, were dominatingly powerful among diverse mammalian lineages. Together with Y gene decay, X-Y arms races molded mammalian sex chromosomes and influenced the course of mammalian evolution.

Mapping Genes Is Good for You.

I abandoned my original career choice of high school teaching to pursue dentistry and soon abandoned that path for genetics. The latter decision was due to a challenge by a professor that led to me reading Nobel speeches by pioneer geneticists before I had formal exposure to the subject. Even then, I was 15 years into my career before my interest in rodent genomes gave way to mapping cattle genes. Events behind these twists and turns in my career path comprise the first part of this review. The remainder is a review of the development of the field of bovine genomics from my personal perspective. I have had the pleasure of working with outstanding graduate students, postdocs, and colleagues to contribute my small part to a discipline that has evolved from a few individuals mapping an orphan genome to a discipline underlying a revolution in animal breeding.

Genomic Structure and Tissue Expression of the NK-Lysin Gene Family in Bison.

Antimicrobial peptides (AMPs) are a class of natural peptides with varying numbers of amino acids. They are principal components of innate immunity in vertebrates, encoding natural antibiotics and providing a protective response against a broad range of microbes including those responsible for tuberculosis, an important disease in bison. NK-lysins are AMPs that have been described in various organisms and are coded by a single gene in several mammalian species, including human. Recently, we described a family of 4 NK-lysin genes in cattle. Here, we examined NK-lysin genes in bison and identified 4 bison paralogs (NK1, NK2A, NK2B, and NK2C), although the current bison genome assembly annotates only 2 (NK1 and NK2). Sequence and phylogenetic analysis support the triplication of NK2 prior to the most recent common ancestor of bison and cattle. Comparative mapping of bison and cattle paralogs indicates that the NK-lysin family is located on bison chromosome 11 with well-conserved synteny of flanking genes relative to cattle. The 3 bison NK-lysin2 genes share high sequence similarity with each other. RNA-seq analysis demonstrates that NK2A, NK2B, and NK2C are expressed primarily in the lung, whereas NK1 is expressed at low levels in all tissues studied. This tissue expression pattern differs from that previously reported for cattle, suggesting some divergence in function since the evolutionary separation of the 2 species.

Duplication of chicken defensin7 gene generated by gene conversion and homologous recombination.

Defensins constitute an evolutionary conserved family of cationic antimicrobial peptides that play a key role in host innate immune responses to infection. Defensin genes generally reside in complex genomic regions that are prone to structural variation, and defensin genes exhibit extensive copy number variation in humans and in other species. Copy number variation of defensin genes was examined in inbred lines of Leghorn and Fayoumi chickens, and a duplication of defensin7 was discovered in the Fayoumi breed. Analysis of junction sequences confirmed the occurrence of a simple tandem duplication of defensin7 with sequence identity at the junction, suggesting nonallelic homologous recombination between defensin7 and defensin6 The duplication event generated two chimeric promoters that are best explained by gene conversion followed by homologous recombination. Expression of defensin7 was not elevated in animals with two genes despite both genes being transcribed in the tissues examined. Computational prediction of promoter regions revealed the presence of several putative transcription factor binding sites generated by the duplication event. These data provide insight into the evolution and possible function of large gene families and specifically, the defensins.

Tissue expression and antibacterial activity of host defense peptides in chicken.

BACKGROUND: Host defence peptides are a diverse group of small, cationic peptides and are important elements of the first line of defense against pathogens in animals. Expression and functional analysis of host defense peptides has been evaluated in chicken but there are no direct, comprehensive comparisons with all gene family and individual genes. RESULTS: We examined the expression patterns of all known cathelicidins, ß-defensins and NK-lysin in multiple selected tissues from chickens. CATH1 through 3 were predominantly expressed in the bone marrow, whereas CATHB1 was predominant in bursa of Fabricius. The tissue specific pattern of ß-defensins generally fell into two groups. ß-defensin1-7 expression was predominantly in bone marrow, whereas ß-defensin8-10 and ß-defensin13 were highly expressed in liver. NK-lysin expression was highest in spleen. We synthesized peptide products of these gene families and analysed their antibacterial efficacy. Most of the host defense peptides showed antibacterial activity against E.coli with dose-dependent efficacy. ß-defensin4 and CATH3 displayed the strongest antibacterial activity among all tested chicken HDPs. Microscopic analyses revealed the killing of bacterium by disrupting membranes with peptide treatment. CONCLUSIONS: These results demonstrate dose-dependent antimicrobial effects of chicken HDPs mediated by membrane damage and demonstrate the differential tissue expression pattern of bioactive HDPs in chicken and the relative antimicrobial potency of the peptides they encode.

Expression of the Bovine NK-Lysin Gene Family and Activity against Respiratory Pathogens.

Unlike the genomes of many mammals that have a single NK-lysin gene, the cattle genome contains a family of four genes, one of which is expressed preferentially in the lung. In this study, we compared the expression of the four bovine NK-lysin genes in healthy animals to animals challenged with pathogens known to be associated with bovine respiratory disease (BRD) using transcriptome sequencing (RNA-seq). The expression of several NK-lysins, especially NK2C, was elevated in challenged relative to control animals. The effects of synthetic peptides corresponding to functional region helices 2 and 3 of each gene product were tested on both model membranes and bio-membranes. Circular dichroism spectroscopy indicated that these peptides adopted a more helical secondary structure upon binding to an anionic model membrane and liposome leakage assays suggested that these peptides disrupt membranes. Bacterial killing assays further confirmed the antimicrobial effects of these peptides on BRD-associated bacteria, including both Pasteurella multocida and Mannhemia haemolytica and an ultrastructural examination of NK-lysin-treated P. multocida cells by transmission electron microscopy revealed the lysis of target membranes. These studies demonstrate that the expanded bovine NK-lysin gene family is potentially important in host defense against pathogens involved in bovine respiratory disease.

Bovine NK-lysin: Copy number variation and functional diversification.

NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ∼30-35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer's patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants.

Mapping a Major Gene for Resistance to Rift Valley Fever Virus in Laboratory Rats.

The Rift Valley Fever virus (RVFV) presents an epidemic and epizootic threat in sub-Saharan Africa, Egypt, and the Arabian Peninsula, and has furthermore recently gained attention as a potential weapon of bioterrorism due to its ability to infect both livestock and humans. Inbred rat strains show similar characteristic responses to the disease as humans and livestock, making them a suitable model species. Previous studies had indicated differences in susceptibility to RVFV hepatic disease among various rat strains, including a higher susceptibility of Wistar-Furth (WF) compared to a more resistant Lewis (LEW) strain. Further study revealed that this resistance trait exhibits the pattern of a major dominant gene inherited in Mendelian fashion. A genome scan of a congenic WF.LEW strain, created from the susceptible WF and resistant LEW strains and itself resistant to infection with RVFV, revealed 2 potential regions for the location of the gene, 1 on chromosome 3 and the other on chromosome 9. Through backcrossing of WF.LEW rats to WF rats, genotyping offspring using SNPs and microsatellites, and viral challenges of 3 N1 litters, we have mapped the gene to the distal end of chromosome 3.

Susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves.

BACKGROUND: Bovine respiratory disease complex (BRDC) is an infectious disease of cattle that is caused by a combination of viral and/or bacterial pathogens. Selection for cattle with reduced susceptibility to respiratory disease would provide a permanent tool for reducing the prevalence of BRDC. The objective of this study was to identify BRDC susceptibility loci in pre-weaned Holstein calves as a prerequisite to using genetic improvement as a tool for decreasing the prevalence of BRDC. High density SNP genotyping with the Illumina BovineHD BeadChip was conducted on 1257 male and 757 female Holstein calves from California (CA), and 767 calves identified as female from New Mexico (NM). Of these, 1382 were classified as BRDC cases, and 1396 were classified as controls, with all phenotypes assigned using the McGuirk health scoring system. During the acquisition of blood for DNA isolation, two deep pharyngeal and one mid-nasal diagnostic swab were obtained from each calf for the identification of bacterial and viral pathogens. Genome-wide association analyses were conducted using four analytical approaches (EIGENSTRAT, EMMAX-GRM, GBLUP and FvR). The most strongly associated SNPs from each individual analysis were ranked and evaluated for concordance. The heritability of susceptibility to BRDC in pre-weaned Holstein calves was estimated. RESULTS: The four statistical approaches produced highly concordant results for 373 top ranked SNPs that defined 126 chromosomal regions for the CA population. Similarly, in NM, 370 SNPs defined 138 genomic regions that were identified by all four approaches. When the two populations were combined (i.e., CA + NM) and analyzed, 324 SNPs defined 116 genomic regions that were associated with BRDC across all analytical methods. Heritability estimates for BRDC were 21% for both CA and NM as individual populations, but declined to 13% when the populations were combined. CONCLUSIONS: Four analytical approaches utilizing both single and multi-marker association methods revealed common genomic regions associated with BRDC susceptibility that can be further characterized and used for genomic selection. Moderate heritability estimates were observed for BRDC susceptibility in pre-weaned Holstein calves, thereby supporting the application of genomic selection to reduce the prevalence of BRDC in U.S. Holsteins.

Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes.

We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism.

Results of the BRD CAP project: progress toward identifying genetic markers associated with BRD susceptibility.

The Bovine Respiratory Disease Coordinated Agricultural Project (BRD CAP) is a 5-year project funded by the United States Department of Agriculture (USDA), with an overriding objective to use the tools of modern genomics to identify cattle that are less susceptible to BRD. To do this, two large genome wide association studies (GWAS) were conducted using a case:control design on preweaned Holstein dairy heifers and beef feedlot cattle. A health scoring system was used to identify BRD cases and controls. Heritability estimates for BRD susceptibility ranged from 19 to 21% in dairy calves to 29.2% in beef cattle when using numerical scores as a semi-quantitative definition of BRD. A GWAS analysis conducted on the dairy calf data showed that single nucleotide polymorphism (SNP) effects explained 20% of the variation in BRD incidence and 17-20% of the variation in clinical signs. These results represent a preliminary analysis of ongoing work to identify loci associated with BRD. Future work includes validation of the chromosomal regions and SNPs that have been identified as important for BRD susceptibility, fine mapping of chromosomes to identify causal SNPs, and integration of predictive markers for BRD susceptibility into genetic tests and national cattle genetic evaluations.

Genome-wide DNA methylation profiles and their relationships with mRNA and the microRNA transcriptome in bovine muscle tissue (Bos taurine).

DNA methylation is a key epigenetic modification in mammals and plays important roles in muscle development. We sampled longissimus dorsi muscle (LDM) from a well-known elite native breed of Chinese Qinchuan cattle living within the same environment but displaying distinct skeletal muscle at the fetal and adult stages. We generated and provided a genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA for fetal and adult muscle studies. Integration analysis revealed a total of 77 and 1,054 negatively correlated genes with methylation in the promoter and gene body regions, respectively, in both the fetal and adult bovine libraries. Furthermore, we identified expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from nine different tissues at multiple developmental stages of bovine muscle-related tissue or organs. In addition, we validated the MeDIP-Seq results by bisulfite sequencing PCR (BSP) in some of the differentially methylated promoters. Together, these results provide valuable data for future biomedical research and genomic and epigenomic studies of bovine skeletal muscle that may help uncover the molecular basis underlying economically valuable traits in cattle. This comprehensive map also provides a solid basis for exploring the epigenetic mechanisms of muscle growth and development.

An update of the goat genome assembly using dense radiation hybrid maps allows detailed analysis of evolutionary rearrangements in Bovidae.

BACKGROUND: The domestic goat (Capra hircus), an important livestock species, belongs to a clade of Ruminantia, Bovidae, together with cattle, buffalo and sheep. The history of genome evolution and chromosomal rearrangements on a small scale in ruminants remain speculative. Recently completed goat genome sequence was released but is still in a draft stage. The draft sequence used a variety of assembly packages, as well as a radiation hybrid (RH) map of chromosome 1 as part of its validation. RESULTS: Using an improved RH mapping pipeline, whole-genome dense maps of 45,953 SNP markers were constructed with statistical confidence measures and the saturated maps provided a fine map resolution of approximate 65 kb. Linking RH maps to the goat sequences showed that the assemblies of scaffolds/super-scaffolds were globally accurate. However, we observed certain flaws linked to the process of anchoring chromosome using conserved synteny with cattle. Chromosome assignments, long-range order, and orientation of the scaffolds were reassessed in an updated genome sequence version. We also present new results exploiting the updated goat genome sequence to understand genomic rearrangements and chromosome evolution between mammals during species radiations. The sequence architecture of rearrangement sites between the goat and cattle genomes presented abundant segmental duplication on regions of goat chromosome 9 and 14, as well as new insertions in homologous cattle genome regions. This complex interplay between duplicated sequences and Robertsonian translocations highlights the rearrangement mechanism of centromeric nonallelic homologous recombination (NAHR) in mammals. We observed that species-specific shifts in ANKRD26 gene duplication are coincident with breakpoint reuse in divergent lineages and this gene family may play a role in chromosome stabilization in chromosome evolution. CONCLUSIONS: We generated dense maps of the complete whole goat genome. The chromosomal maps allowed us to anchor and orientate assembled genome scaffolds along the chromosomes, annotate chromosome rearrangements and thereby get a better understanding of the genome evolution of ruminants and other mammals.

Mapping and genotypic analysis of the NK-lysin gene in chicken.

BACKGROUND: Antimicrobial peptides (AMP) are important elements of the first line of defence against pathogens in animals. NK-lysin is a cationic AMP that plays a critical role in innate immunity. The chicken NK-lysin gene has been cloned and its antimicrobial and anticancer activity has been described but its location in the chicken genome remains unknown. Here, we mapped the NK-lysin gene and examined the distribution of a functionally significant single nucleotide polymorphism (SNP) among different chicken inbred lines and heritage breeds. RESULTS: A 6000 rad radiation hybrid panel (ChickRH6) was used to map the NK-lysin gene to the distal end of chromosome 22. Two additional genes, the adipocyte enhancer-binding protein 1-like gene (AEBP1) and the DNA polymerase delta subunit 2-like (POLD2) gene, are located in the same NW_003779909 contig as NK-lysin, and were thus indirectly mapped to chromosome 22 as well. Previously, we reported a functionally significant SNP at position 271 of the NK-lysin coding sequence in two different chicken breeds. Here, we examined this SNP and found that the A allele appears to be more common than the G allele in these heritage breeds and inbred lines. CONCLUSIONS: The chicken NK-lysin gene mapped to the distal end of chromosome 22. Two additional genes, AEBP1 and POLD2, were indirectly mapped to chromosome 22 also. SNP analyses revealed that the A allele, which encodes a peptide with a higher antimicrobial activity, is more common than the G allele in our tested inbred lines and heritage breeds.

Chicken NK-lysin is an alpha-helical cationic peptide that exerts its antibacterial activity through damage of bacterial cell membranes.

The antimicrobial peptides (AMP) are important elements of the first line of defense against pathogens in animals, and an important constituent of innate immunity. Antimicrobial peptides act on a broad spectrum of microbial organisms. NK-Lysin is a cationic antibacterial peptide that was originally isolated from porcine intestinal tissue based on its antibacterial activity. We synthesized peptides corresponding to each helical region of chicken NK-lysin and analyzed their secondary structures in addition to their antimicrobial activity. Circular dichroism spectroscopy of the synthetic chicken NK-lysin (cNK-78) and 4 small peptides in negatively charged liposomes demonstrated transition in the conformation of α-helical peptides relative to the charged environment. Chicken NK-lysin inhibits the growth of a representative gram-negative bacterium, Escherichia coli. The antimicrobial activity of 2 peptides designated H23 and H34 was similar to that of mature NK-lysin, cNK-78. Microscopic analyses revealed the death of bacterium with disrupted membranes after peptide treatment, suggesting that chicken NK-lysin, an alpha-helical cationic peptide, exerts its antimicrobial activity by damaging the bacterial cell membrane.

Functional characterization of naturally occurring melittin peptide isoforms in two honey bee species, Apis mellifera and Apis cerana.

Insect-derived antimicrobial peptides (AMPs) have diverse effects on antimicrobial properties and pharmacological activities such as anti-inflammation and anticancer properties. Naturally occurring genetic polymorphism have a direct and/or indirect influence on pharmacological effect of AMPs, therefore information on single nucleotide polymorphism (SNP) occurring in natural AMPs provides an important clue to therapeutic applications. Here we identified nucleotide polymorphisms in melittin gene of honey bee populations, which is one of the potent AMP in bee venoms. We found that the novel SNP of melittin gene exists in these two honey bee species, Apis mellifera and Apis cerana. Nine polymorphisms were identified within the coding region of the melittin gene, of which one polymorphism that resulted in serine (Ser) to asparagine (Asp) substitution that can potentially effect on biological activities of melittin peptide. Serine-substituted melittin (Mel-S) showed more cytotoxic effect than asparagine-substituted melittin (Mel-N) against E. coli. Also, Mel-N and Mel-S had different inhibitory effects on the production of inflammatory factors such as IL-6 and TNF-α in BV-2 cells. Moreover, Mel-S showed stronger cytotoxic activities than Mel-N peptide against two human ovarian cancer cell lines. Using carbon nanotube-based transistor, we here characterized that Mel-S interacted with small unilamellar liposomes more strongly than Mel-N. Taken together, our present study demonstrates that there exist different characteristics of the gene frequency and the biological activities of the melittin peptide in two honey bee species, Apis mellifera and A. cerana.

Gene expression and DNA methylation status of chicken primordial germ cells.

DNA methylation reprogramming of primordial germ cells (PGCs) in mammals establishes monoallelic expression of imprinting genes, maintains retrotransposons in an inactive state, inactivates one of the two X chromosomes, and suppresses gene expression. However, the roles of DNA methylation in chickens PGCs are unknown. In this study, we found a 1.5-fold or greater difference in the expression of 261 transcripts when comparing PGCs and chicken embryonic fibroblasts (CEFs) using an Affymetrix GeneChip Chicken Genome Array. In addition, we analyzed the methylation patterns of the regions ~5-kb upstream of 261 sorted genes, 51 of which were imprinting homologous loci and 49 of which were X-linked homologous loci in chicken using the MeDIP Array by Roche NimbleGen. Seven hypomethylated and five hypermethylated regions within the 5-kb upstream regions of 261 genes were found in PGCs when compared with CEFs. These differentially methylated regions were restrictively matched to differentially expressed genes in PGCs. We also detected 203 differentially methylated regions within imprinting and X-linked homologous regions between male PGCs and female PGCs. These differentially methylated regions may be directly or indirectly associated with gene expression during early embryonic development, and the epigenetic difference could be evolutionally conserved between mammals and birds.

Genomics of complex traits.

The analysis of complex genetic traits, including mapping and identification of causative genes, has long been an enigma of genetic biology, whether in the animal sciences or in medical sciences. Traits of agricultural interest and traits of medical interest are often under the influence of both environmental factors and multiple genes, each with modest contributions to the total variance in the trait. Although the number of known mutations underlying complex traits is still relatively small, advances in genomics have greatly enhanced traditional pathways to their analysis and gene mining. The candidate gene approach, linkage analysis, and association studies are all significantly more powerful with recent advances in genome mapping, sequencing, and analysis of individual variation. Avenues to gene discovery are discussed with emphasis on genome wide association studies (GWAS) and the use of single nucleotide polymorphisms (SNPs) as revealed by increasingly powerful commercially available microarrays.

Effects of a single nucleotide polymorphism in the chicken NK-lysin gene on antimicrobial activity and cytotoxicity of cancer cells.

NK-lysin is an effector protein of the innate immune system and an important component of host protection. We isolated a SNP in the NK-lysin coding sequence among different chicken breeds. This A to G substitution at the position 271 nucleotide in the ORF results in an Asn (N) to Asp (D) amino acid alteration. We synthesized two 30-aa peptides (N29N and N29D) to compare the biological activity of the helix 2-loop-helix 3 region of NK-lysin resulting from the polymorphic gene. Both peptides were found to be cytotoxic in bacteria and tumor cell cultures at micromolar concentrations. The N29N peptide, however, exhibited greater antibacterial and anticancer activity than the N29D peptide. Circular dichroism spectroscopy of the two peptides in negatively charged single unilamellar vesicles showed spectra typical of α-helical peptides. The helical profile of N29D was reduced substantially compared with that of N29N. However, no structural change was observed in neutral vesicles. ζ-Potential measurements of liposomes incubated with increasing peptide concentrations allowed surface charge neutralization with a negatively charged lipid, but not with a zwitterionic lipid. This result suggests that a difference in electrostatic interaction between lipid membranes and the helical peptides results from the polymorphic gene and is subsequently an important factor in cell lytic activity of variant NK-lysin peptides.

First steps: bovine genomics in historical perspective.

The vision of Morris Soller was instrumental in launching the field of bovine genomics. This study is a review of the early years of bovine gene mapping leading up to the sequencing and assembly of the bovine genome in 2009. A historical perspective of parasexual, linkage and physical mapping is provided with a focus on the contribution of these maps to the eventual assignment and orientation of genes and sequence to cattle chromosomes.